Kimio Iwano

Cloning of the xynNB Gene Encoding Xylanase B from Aspergillus niger and Its Expression in Aspergillus kawachii

Abstract Aspergillus niger IFO 4066 produced two xylanases, xylanase A (XynNA) and xylanase B (XynNB), in culture medium, and these enzymes were purified. Acidophilic xylanase such as xylanase C (XynC) of white koji mold (Aspergillus kawachii IFO 4308) was not detected in A. niger cultures. However, results of Southern analysis using xynC cDNA of A. kawachii as a probe suggested that A. niger contained a gene homologous to xynC of A. kawachii. Therefore, we cloned this xylanase gene from A. niger. The predicted amino acid sequence of the cloned xylanase showed a homology to that of xynC of A. kawachii. However, a large number of amino acid substitutions were detected, especially in the N-terminal region. Both this cloned gene and xynC gene of A. kawachii had an intron at the same position in the coding region. The cloned gene was expressed in A. kawachii and a large quantity of xylanase was produced. The elution profile on an anion exchange chromatogram and the N-terminal amino acid sequence of the xylanase purified from the transformant were the same as those of XynNB. This confirmed that the cloned gene encoded XynNB.

Conversion of ferulic acid into 4-vinylguaiacol, vanillin and vanillic acid in model solutions of shochu

4-Vinylguaiacol (4-hydroxy-3-methoxystyrene), vanillin (4-hydroxy-3-methoxybenzaldehyde), and vanillic acid (4-hydroxy-3-methoxybenzoic acid) were isolated from distilled and stored model solutions of shochu (MSS) that originally contained only ferulic acid (4-hydroxy-3-methoxycinnamic acid) using a solid-phase extraction technique. These compounds were analyzed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The amounts of the metabolites converted from ferulic acid was affected by pH, alcohol concentration, and temperature of during storage. The identities of the metabolites were tentatively determined by gas chromatography-mass spectrometry (GC-MS). The results of this experiment suggested that vanillin was formed via 4-vinylguaiacol from ferulic acid, and was then converted to vanillic acid. The pathway of the decarboxylation of ferulic acid appeared to be one of the chemical transformation.

Role of Two α-l-arabinofuranosidases in arabinoxylan degradation and characteristics of the encoding genes from shochu koji molds, Aspergillus kawachii and Aspergillus awamori

Two different alpha-L-arabinofuranosidases from Aspergillus kawachii were purified and characterized. The two enzymes acted synergically with xylanase in the degradation of arabinoxylan and resulted in an increase in the amount of ferulic acid release by feruloyl esterase. Both enzymes were acidophilic and acid stable enzymes which had an optimum pH of 4.0 and were stable at pH 3.0-7.0. The general properties of the enzymes including pH optima and pH stability were similar to those of Aspergillus awamori. These results suggest that the alpha-L-arabinofuranosidases contribute to an increase in cereal utilization and formation of aroma in shochu brewing. Two different genes encoding alpha-L-arabinofuranosidases from A. kawachii, designated as AkabfA and AkabjB, and those from A. awamori, designated as AwabfA and AwabjB, were also cloned and characterized. The difference between the sequences of AkabfA and AwabfA was only one nucleotide, resulting in an amino acid difference in the sequence, and the enzymes were assigned to family 51 of glycoside hydrolases. On the other hand, the differences between the sequences of AkabjB and AwabjB and between their encoding proteins were two nucleotides and one amino acid residue, respectively, and the enzymes were assigned to family 54 of glycoside hydrolases. On comparison of the abfA and abjB genes among A. kawachii, A. awamori, and A. niger, the relationship between the two genes for A. kawachii and A. awamori was much closer than those between A. niger and the others. Northern analyses showed that transcription of AkabfB was greater than that of AkabfA in the presence of L-arabitol and L-arabinose, and that transcriptions of both genes were not induced in the presence of sucrose and glucose.

Efficient selection of hybrids by protoplast fusion using drug resistance markers and reporter genes in Saccharomyces cerevisiae

We have developed a selection system for hybrids by protoplast fusion using dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A, and reporter genes, ADH1p-PHO5-ADH1t and CLN2p-CYC1-lacZ, in Saccharomyces cerevisiae. To examine the effectiveness of this system, plasmids with each marker and reporter gene were introduced into auxotrophic sake yeasts. From the resulting transformants, eight colonies were screened by protoplast fusion in combination with the drug resistance markers and the reporter genes. Among them, seven strains were judged as hybrids between parental strains by analysis of growth on a minimal medium. This selection system was applied to wine yeasts having no genetic markers. Six strains were regarded as hybrids between parental strains by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene and by karyotype analysis using a contour-clamped homogeneous electric field (CHEF). We propose that the plotoplast fusion using dominant selective geneticin- and aureobasidin A-resistance markers and reporter genes is useful for the selection of hybrids from wine yeasts, which are homothallic and have low sporulation ability.

Cln3 blocks IME1 transcription and the Ime1-Ume6 interaction to cause the sporulation incompetence in a sake yeast, Kyokai no. 7

Industrial yeasts, including a sake yeast Kyokai no. 7 (K7), are generally unable to sporulate. In K7 (Saccharomyces cerevisiae) cells, IME1 transcription was not induced under sporulation conditions, and K7 cells partially restored sporulation ability when transformed with a multicopy plasmid bearing IME1. However, the mechanisms of sporulation incompetence in industrial yeasts are poorly understood. We demonstrated that the deletion of the G1 cyclin CLN3, a key activator of the cell cycle, allows K7 cells to induce IME1 transcription and sporulate under sporulation conditions. In K7 cells, CLN3 mRNA and protein were not down-regulated despite sporulation conditions. Moreover, using a two-hybrid assay, we found that Ime1-Ume6 interaction was promoted in Cln3-deficient K7 cells. Thus, Cln3 is involved in the mechanism underlying sporulation incompetence by inhibiting IME1 transcription and the Ime1-Ume6 interaction. Based on these findings, we hypothesize that the absence of transmission of nutrient starvation signals to CLN3 leads to sporulation incompetence in K7 cells.